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On photolithographic oligonucleotide arrays, in contrast, multiple probes from the same single
gene of interest, each with a corresponding mismatch probe that serves as internal control, as
well as known amounts of labeled transcripts for genes that serve as internal standards, are
hybridized simultaneously to the microarray to enable quantitation of transcript abundance.
Gene expression experiments can also be performed by hybridizing a single labeled
mRNA sample to “macroarrays” of DNA elements that are supported on positively charged
filters. Specialty arrays can be made and analyzed by this method relatively cheaply, and human,
mouse, and microbial macroarrays are commercially available (SigmaGenosys, The Woodlands,
TX; Research Genetics, Huntsville, AL; Clontech Laboratories, Palo Alto, CA; Genome
Systems, St. Louis, MO). The major disadvantages of this format are reduced sensitivity, limited
numbers of elements, and the need for higher concentrations of labeled cDNA (12).
Microarray technology is sufficiently promising for medical and pharmaceutical
applications that it is expected to continue to attract strong commercial interest leading to
increasing array element density, greater detection sensitivity, and more cost-effective methods.
Additional details and technical descriptions are available in recent reviews (14, 15).
2.5 Mutagenesis
The DNA molecule consists of two strands of nucleotide bases held together by hydrogen
bonds between complementary bases: adenosine pairs with thymine, and cytosine pairs with
guanine. Within the coding region of a gene, each triplet of nucleotides specifies an amino acid,
and the strand of amino acids in turn folds, sometimes after processing, into a functional protein.
DNA is replicated during each cell division in a growing organism, and although this process is
highly accurate, errors can nevertheless occur in which an incorrect nucleotide is inserted into
the daughter strand. If the incorrect nucleotide is copied faithfully in subsequent replication, a
mutation is generated that may ultimately affect the function of the resulting protein and give rise
to a variant organism (16).
Mutations arise in nature either randomly or as consequences of DNA damage by
ultraviolet (UV) radiation, chemicals, or other mutagenic agents (16). Researchers seeking
desirable mutations can accelerate the mutagenic process by four primary methods: stimulating
replication error-based mutagenesis with chemicals or radiation; generating localized sequence
alterations through error-prone PCR; inducing combinatorial mutagenesis through directed
evolution; or using structural information about a protein to change specific amino acids, termed
“rational design.” Each of the latter three is conducted in vitro and must therefore be followed
by reintroduction of the mutated sequence into the host organism. If desired mutations show
phenotypes that are recessive to the wild-type phenotype, then the wild-type gene must first be
disabled or displaced by integration of the new sequence in its place; often, however, the desired
mutations have dominant phenotypes (e.g., enabling function under harsh conditions) so that
such measures are not necessary. Nevertheless, expression of a mutant sequence can be
vulnerable to the transcriptional environment at its locus of integration. In the case that the
mutant sequence must be integrated into host DNA to be maintained stably, numerous
transfections may be required before satisfactory expression is obtained.
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